Dear Partha Treat your culture with Kifunensine prior to transfection (or throughout growth if you're using stables) and then treat purified protein with EndoH. Pretty cheap and effective.
PNGase F does not *require* denaturing conditions. It just likes the protein to be 'loose' - in my experience about half the time the accessible sites will fall off even under native conditons. However, PNGase F is somewhat expensive and after treatment certain proteins have fallen out of solution (presumably because not a single carbohydrate remained, as opposed to 'shielding' provided by the single remaining sugar left behind by EndoH). Caveat emptor. Artem - Cosmic Cats approve of this message On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar < spart...@gmail.com> wrote: > Dear All, > > I am in a situation, almost for the first time within my limited > experience, that deglycosylation might be necessary to obtain crystal. So, > I thought of tapping to vast experience of CCP4BBers, while I am searching > literature. > > I have protein that has been expressed in HEK293 cells, secreted into > media, purified over IMAC and SEC columns. Crystallization-screens with its > binding partners (they form good complexes based on analytical SEC) have > not produced any useful hits (whereas complexes with related proteins > worked well). So, I plan to re-try complex formation and \crystallization > screen after deglysosylation. > > My question is: In practice, Does a kit (for example here: https://www. > sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en®ion=US) > containing Endo F1, F2, F3 be sufficient or should this be tried in > combination with PNGase (which requires > desaturating conditions)?!! > > Many Thanks in advance for your suggestions, and reference. > > Best Wishes, > Partha > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
